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Genome-wide profiling of the 3' ends of polyadenylated RNAs.

ABSTRACT: Alternative polyadenylation (APA) diversifies the 3' termini of a majority of mRNAs in most eukaryotes, and is consequently inferred to have substantial consequences for the utilization of post-transcriptional regulatory mechanisms. Since conventional RNA-sequencing methods do not accurately define mRNA termini, a number of protocols have been developed that permit sequencing of the 3' ends of polyadenylated transcripts (3'-seq). We present here our experimental protocol to generate 3'-seq libraries using a dT-priming approach, including extensive details on considerations that will enable successful library cloning. We pair this with a set of computational tools that allow the user to process the raw sequence data into a filtered set of clusters that represent high-confidence functional polyadenylation sites. The data are single-nucleotide resolution and quantitative, and can be used for downstream analyses of APA.

SUBMITTER: Sanfilippo P

PROVIDER: S-EPMC5583017 | biostudies-literature | 2017 Aug

REPOSITORIES: biostudies-literature

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Genome-wide profiling of the 3' ends of polyadenylated RNAs.

Sanfilippo Piero P Miura Pedro P Lai Eric C EC

Methods (San Diego, Calif.) 20170608

Alternative polyadenylation (APA) diversifies the 3' termini of a majority of mRNAs in most eukaryotes, and is consequently inferred to have substantial consequences for the utilization of post-transcriptional regulatory mechanisms. Since conventional RNA-sequencing methods do not accurately define mRNA termini, a number of protocols have been developed that permit sequencing of the 3' ends of polyadenylated transcripts (3'-seq). We present here our experimental protocol to generate 3'-seq libra ...[more]

PMID: 28602807

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